The present invention relates to an improved method for determining nucleotide sequences on biochips/micro-assays, for example, using p53 mediated hybridization. The present invention relates particularly to the use of p53 polypeptides in determining changes in nucleotide sequences.
On the basis of data provided by the Human Genome Project (HUGO) and the findings regarding the genomic sequences of other organisms such as bacteria, increasing use of biochips or micro-arrays have been utilized for demonstration or investigation of such nucleotide sequences and, at the same time, a large number of different nucleotide sequences have been minutely tested.
Nucleotides of defined lengths or different sequences have been applied at pre-defined sites of a carrier and the carrier is then brought in contact with second nucleotide linked to an identifiable marker under conditions enabling binding of the second nucleotide preferentially to a nucleotide complementary thereto. After removal of non-hybridized nucleotides, detection of the marker at a defined position of the biochip or micro-array is an indication of the presence or absence of a property to be investigated.
As regards the current sequence data, it was found that certain characteristics of higher organisms, such as the tendency towards development of certain diseases or, in the case of the bacteria, the development of resistance to antibiotics, are based on a change only in a small number and in some cases only a single nucleotide (“single nucleotide polymorphism,” SNP) in the sequence of the respective gene responsible therefore or involved therein. Therefore, there is an increased need for determining or demonstrating such changes in the genome of organisms in order to conduct, for example, population studies or forensic medical examinations or in order to demonstrate the presence of antibiotic-resistant bacteria.
At the present time, such minute changes in nucleotide sequences can be detected by means of various methods such as, for example, sequencing the nucleotide sequences of interest, restriction fragment length polymorphism (RFLP) analyses or allele-specific hybridization using longer probe sequences. Such methods, however, because of the inherent methodological and time expenditures in their performance, are unsuitable for screening of larger numbers of samples.
Although the use of biochips or micro-arrays in association with simple hybridization techniques would be desirable, the problem of specificity has not been satisfactorily resolved to date. In practice, it has been found to be extremely difficult to avoid selection of the probe-nucleotide or hybridization conditions so that hybridization of the probe-nucleotide using probe DNA that does not have a complementary nucleotide at the concerned site, i.e. the site to be investigated.
In order to solve these known problems of the prior art, the so-called “single nucleotide primer extension” method has been proposed. In this method, a short nucleotide sequence (the primer) is hybridized on the DNA to be investigated, which is applied onto a biochip/micro-array and then extended by one nucleotide using a DNA polymerase. An extension will occur in this procedure only if there is compatibility between the deoyxynucleotide triphospate presented with the complementary strand. The eventually elongated primers can be separated on the basis of their length on a gel or using their size with by means of MALDI (matrix-assisted laser desorption/ionization mass spectrometry). According to an alternative embodiment, the deoxynucleotide triphosphate presented can be provided with a marker, so that the presence of the marker can be correlated directly with the presence of a particular nucleotide in the complementary strand.
Although this method eliminates several of the known problems, it is still costly, because it is a two-step procedure, and ultimately is limited to the determination of the presence of a single nucleotide change in a DNA sequence.
Thus, there continues to be a need for a method, by which a small number or even only a single nucleotide change in a nucleotide sequence can be determined.